This is a broad proposal for the study of 3 folate mediated enzymes important to cell growth: dihydrofolate reductase, thymidylate synthetase, polyglutamate synthetase. The enzymes will be characterized and studied from mammalian tissues, and the regulation of these enzymes will be studied using mouse and human neoplastic cells propagated in vitro. The interaction of known inhibitors of these enzymes will be characterized: fluorouracil (FdUMP) and methotrexate. The mechanisms of resistance to methotrexate in vitro, and in patients receiving this chemotherapeutic agent will be investigated utilizing highly sensitive and specific methodology, and the techniques of recombinant DNA cloning. New inhibitors of the three enzymes will be synthesized and evaluated.